PNH testing methodologies

High-sensitivity flow cytometry

High-sensitivity flow cytometry is the current ‘gold standard’ diagnostic test for paroxysmal nocturnal haemoglobinuria (PNH) as it provides the most quantitative and qualitative information.1,2 Using a combination of specific antibodies, fluorescent aerolysin (FLAER) and flow cytometry gating strategies, the presence and size of a PNH clone can be determined.3-7 Monoclonal antibodies, such as anti-CD59, bind to the glycosylphosphatidylinositol (GPI)-linked protein, whereas FLAER, a fluorescently labelled, inactive bacterial toxin, targets the GPI anchor on the cell surface directly, selectively revealing the presence or absence of GPI-anchored proteins.8

High-sensitivity flow cytometry is the current ‘gold standard’ diagnostic test for PNH

Flow cytometry provides a direct measure of the expression of GPI anchor and GPI-linked proteins (such as CD59) on all haematopoietic cell populations, allowing identification of three distinct PNH clonal populations:3,9

Type I cells: normal expression of GPI-anchored proteins

Type II cells: intermediate expression of GPI-anchored proteins

Type III cells: no detectable expression of GPI-anchored proteins

As PNH is a rare condition (up to 15.9 cases/million),10 many laboratories have never seen a positive case. Variability in testing can be introduced at every step of the process, including sample preparation, antibody selection, instrument set-up, gating strategy, interpretation of results, selection of suitable quality controls and reporting of results. To avoid inter- and intra-laboratory variations in testing procedures, specialised expertise in flow cytometry is required in combination with standardised testing procedures. Further information and guidance on high-quality PNH testing can be found in PNH testing guidelines.

Genetic testing

Recently, mutations of the phosphatidylinositol glycan class A gene, PIG-A, have been identified as the underlying genetic cause of PNH.11 Genetic sequencing of the PIG-A gene could be used to confirm the presence of PNH clones following flow cytometry, but should not be applied to routine testing as >100 mutations of the PIG-A gene have been identified12 with no clear association to PNH subtype or disease severity.

Genetic testing should not be used for diagnosis of, or routine testing for, PNH